Optimized delivery of RNA silencing prolyl hydroxylase domain 2 for enhanced angiogenesis and osteogenesis using bioactive glass scaffolds

Abstract

This study investigated the delivery of small interfering RNA (siRNA) silencing prolyl hydroxylase domain 2 (PHD2) via poly(lactic-co-glycolic acid) (PLGA)-bioactive glass scaffold to improve angiogenesis and osteogenesis for biomineralized tissue engineering. Polyethylenimine (PEI) was complexed with PHD2siRNA at 3:1, 6:1, and 8:1 ratios, and the resulting complexes were characterized by transmission electron microscopy, NanoSight analysis, and gel retardation assays. Their biological effects on human umbilical vein endothelial cells (HUVECs) were evaluated to determine the optimal ratio. PLGA-bioactive glass scaffolds were prepared and loaded with the most effective PEI/PHD2siRNA complex. HUVECs were used to confirm controlled RNA release from the scaffolds. The angiogenic effects of HUVECs and osteogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) were evaluated. The 8:1 ratio of PEI/PHD2siRNA demonstrated the highest efficiency, improving HUVEC tube formation. Incorporating these complexes into PLGA-bioactive glass scaffolds confirmed RNA release, as indicated by improved tube formation. The scaffolds promoted the proliferation and angiogenic differentiation of HUVECs. SHED cells cultured on the scaffolds demonstrated improved proliferation and osteogenic differentiation, evidenced by elevated alkaline phosphatase activity and mineral deposition. Coculturing SHED cells with HUVECs on the scaffolds exerted synergistic effects, producing more robust osteogenic differentiation than culturing SHED cells alone. Immunofluorescence staining revealed increased expression of both angiogenic and osteogenic markers. PHD2 siRNA delivery via PLGA-bioactive glass scaffolds enhanced angiogenesis and osteogenesis, supporting its potential in tissue engineering. This dual-function scaffold offers a promising strategy for dental and craniofacial regeneration.